Impact of Herpes Simplex Virus Type 2 and Human Immunodeficiency Virus Dual Infection on Female Genital Tract Mucosal Immunity and the Vaginal Microbiome.

BACKGROUND: Mechanisms linking herpes simplex virus type 2 (HSV-2) with human immunodeficiency virus (HIV) are not fully defined. We tested the hypothesis that HSV-2 and HIV dual infection is associated with cervicovaginal inflammation and/or vaginal dysbiosis. METHODS: Genital tract samples were obtained weekly over a 12-week period from 30 women seropositive (+) for HIV and HSV-2 and 15 women each who were seropositive for one or seronegative (-) for both viruses. Immune mediators, antimicrobial activity, and microbial composition and diversity were compared. RESULTS: Significant differences in the concentrations of interferon-γ (P = .002), tumor necrosis factor-α (P = .03), human beta defensin 1 (P = .001), secretory leukocyte protease inhibitor (P = .01), and lysozyme (P = .03) were observed across the 4 groups (Kruskal-Wallis). There were also significant differences in vaginal microbial alpha diversity (Simpson index) (P = .0046). Specifically, when comparing HIV-1+/HSV-2+ to HIV-1-/HSV-2- women, a decrease in Lactobacillus crispatus and increase in diverse anaerobes was observed. The number of genital HSV outbreaks was greater in HIV+ versus HIV- women (39 versus 12) (P = .04), but there were no significant differences when comparing outbreak to non-outbreak visits. CONCLUSIONS: Increased microbial diversity and cervicovaginal inflammation in HIV and HSV-2 dually infected women may adversely impact genital health and, in the absence of antiretroviral therapy, facilitate HIV shedding.

Does the human placenta delivered at term have a microbiota? Results of cultivation, quantitative real-time PCR, 16S rRNA gene sequencing, and metagenomics.

BACKGROUND: The human placenta has been traditionally viewed as sterile, and microbial invasion of this organ has been associated with adverse pregnancy outcomes. Yet, recent studies that utilized sequencing techniques reported that the human placenta at term contains a unique microbiota. These conclusions are largely based on the results derived from the sequencing of placental samples. However, such an approach carries the risk of capturing background-contaminating DNA (from DNA extraction kits, polymerase chain reaction reagents, and laboratory environments) when low microbial biomass samples are studied. OBJECTIVE: To determine whether the human placenta delivered at term in patients without labor who undergo cesarean delivery harbors a resident microbiota ("the assemblage of microorganisms present in a defined niche or environment"). STUDY DESIGN: This cross-sectional study included placentas from 29 women who had a cesarean delivery without labor at term. The study also included technical controls to account for potential background-contaminating DNA, inclusive in DNA extraction kits, polymerase chain reaction reagents, and laboratory environments. Bacterial profiles of placental tissues and background technical controls were characterized and compared with the use of bacterial culture, quantitative real-time polymerase chain reaction, 16S ribosomal RNA gene sequencing, and metagenomic surveys. RESULTS: (1) Twenty-eight of 29 placental tissues had a negative culture for microorganisms. The microorganisms retrieved by culture from the remaining sample were likely contaminants because corresponding 16S ribosomal RNA genes were not detected in the same sample. (2) Quantitative real-time polymerase chain reaction did not indicate greater abundances of bacterial 16S ribosomal RNA genes in placental tissues than in technical controls. Therefore, there was no evidence of the presence of microorganisms above background contamination from reagents in the placentas. (3) 16S ribosomal RNA gene sequencing did not reveal consistent differences in the composition or structure of bacterial profiles between placental samples and background technical controls. (4) Most of the bacterial sequences obtained from metagenomic surveys of placental tissues were from cyanobacteria, aquatic bacteria, or plant pathogens, which are microbes unlikely to populate the human placenta. Coprobacillus, which constituted 30.5% of the bacterial sequences obtained through metagenomic sequencing of placental samples, was not identified in any of the 16S ribosomal RNA gene surveys of these samples. These observations cast doubt as to whether this organism is really present in the placenta of patients at term not in labor. CONCLUSION: With the use of multiple modes of microbiologic inquiry, a resident microbiota could not be identified in human placentas delivered at term from women without labor. A consistently significant difference in the abundance and/or presence of a microbiota between placental tissue and background technical controls could not be found. All cultures of placental tissue, except 1, did not yield bacteria. Incorporating technical controls for potential sources of background-contaminating DNA for studies of low microbial biomass samples, such as the placenta, is necessary to derive reliable conclusions.

Genomic comparison of Trypanosoma conorhini and Trypanosoma rangeli to Trypanosoma cruzi strains of high and low virulence.

BACKGROUND: Trypanosoma conorhini and Trypanosoma rangeli, like Trypanosoma cruzi, are kinetoplastid protist parasites of mammals displaying divergent hosts, geographic ranges and lifestyles. Largely nonpathogenic T. rangeli and T. conorhini represent clades that are phylogenetically closely related to the T. cruzi and T. cruzi-like taxa and provide insights into the evolution of pathogenicity in those parasites. T. rangeli, like T. cruzi is endemic in many Latin American countries, whereas T. conorhini is tropicopolitan. T. rangeli and T. conorhini are exclusively extracellular, while T. cruzi has an intracellular stage in the mammalian host. RESULTS: Here we provide the first comprehensive sequence analysis of T. rangeli AM80 and T. conorhini 025E, and provide a comparison of their genomes to those of T. cruzi G and T. cruzi CL, respectively members of T. cruzi lineages TcI and TcVI. We report de novo assembled genome sequences of the low-virulent T. cruzi G, T. rangeli AM80, and T. conorhini 025E ranging from ~ 21-25 Mbp, with ~ 10,000 to 13,000 genes, and for the highly virulent and hybrid T. cruzi CL we present a ~ 65 Mbp in-house assembled haplotyped genome with ~ 12,500 genes per haplotype. Single copy orthologs of the two T. cruzi strains exhibited ~ 97% amino acid identity, and ~ 78% identity to proteins of T. rangeli or T. conorhini. Proteins of the latter two organisms exhibited ~ 84% identity. T. cruzi CL exhibited the highest heterozygosity. T. rangeli and T. conorhini displayed greater metabolic capabilities for utilization of complex carbohydrates, and contained fewer retrotransposons and multigene family copies, i.e. trans-sialidases, mucins, DGF-1, and MASP, compared to T. cruzi. CONCLUSIONS: Our analyses of the T. rangeli and T. conorhini genomes closely reflected their phylogenetic proximity to the T. cruzi clade, and were largely consistent with their divergent life cycles. Our results provide a greater context for understanding the life cycles, host range expansion, immunity evasion, and pathogenesis of these trypanosomatids.

The Evolutionary Loss of RNAi Key Determinants in Kinetoplastids as a Multiple Sporadic Phenomenon.

We screened the genomes of a broad panel of kinetoplastid protists for genes encoding proteins associated with the RNA interference (RNAi) system using probes from the Argonaute (AGO1), Dicer1 (DCL1), and Dicer2 (DCL2) genes of Leishmania brasiliensis and Crithidia fasciculata. We identified homologs for all the three of these genes in the genomes of a subset of these organisms. However, several of these organisms lacked evidence for any of these genes, while others lacked only DCL2. The open reading frames encoding these putative proteins were structurally analyzed in silico. The alignments indicated that the genes are homologous with a high degree of confidence, and three-dimensional structural models strongly supported a functional relationship to previously characterized AGO1, DCL1, and DCL2 proteins. Phylogenetic analysis of these putative proteins showed that these genes, when present, evolved in parallel with other nuclear genes, arguing that the RNAi system genes share a common progenitor, likely across all Kinetoplastea. In addition, the genome segments bearing these genes are highly conserved and syntenic, even among those taxa in which they are absent. However, taxa in which these genes are apparently absent represent several widely divergent branches of kinetoplastids, arguing that these genes were independently lost at least six times in the evolutionary history of these organisms. The mechanisms responsible for the apparent coordinate loss of these RNAi system genes independently in several lineages of kinetoplastids, while being maintained in other related lineages, are currently unknown.

Changes in vaginal community state types reflect major shifts in the microbiome.

Background: Recent studies of various human microbiome habitats have revealed thousands of bacterial species and the existence of large variation in communities of microorganisms in the same habitats across individual human subjects. Previous efforts to summarize this diversity, notably in the human gut and vagina, have categorized microbiome profiles by clustering them into community state types (CSTs). The functional relevance of specific CSTs has not been established. Objective: We investigate whether CSTs can be used to assess dynamics in the microbiome. Design: We conduct a re-analysis of five sequencing-based microbiome surveys derived from vaginal samples with repeated measures. Results: We observe that detection of a CST transition is largely insensitive to choices in methods for normalization or clustering. We find that healthy subjects persist in a CST for two to three weeks or more on average, while those with evidence of dysbiosis tend to change more often. Changes in CST can be gradual or occur over less than one day. Upcoming CST changes and switches to high-risk CSTs can be predicted with high accuracy in certain scenarios. Finally, we observe that presence of Gardnerella vaginalis is a strong predictor of an upcoming CST change. Conclusion: Overall, our results show that the CST concept is useful for studying microbiome dynamics.

MeFiT: merging and filtering tool for illumina paired-end reads for 16S rRNA amplicon sequencing.

BACKGROUND: Recent advances in next-generation sequencing have revolutionized genomic research. 16S rRNA amplicon sequencing using paired-end sequencing on the MiSeq platform from Illumina, Inc., is being used to characterize the composition and dynamics of extremely complex/diverse microbial communities. For this analysis on the Illumina platform, merging and quality filtering of paired-end reads are essential first steps in data analysis to ensure the accuracy and reliability of downstream analysis. RESULTS: We have developed the Merging and Filtering Tool (MeFiT) to combine these pre-processing steps into one simple, intuitive pipeline. MeFiT invokes CASPER (context-aware scheme for paired-end reads) for merging paired-end reads and provides users the option to quality filter the reads using the traditional average Q-score metric or using a maximum expected error cut-off threshold. CONCLUSIONS: MeFiT provides an open-source solution that permits users to merge and filter paired end illumina reads. The tool has been implemented in python and the source-code is freely available at https://github.com/nisheth/MeFiT .

Dynamical System Modeling to Simulate Donor T Cell Response to Whole Exome Sequencing-Derived Recipient Peptides Demonstrates Different Alloreactivity Potential in HLA-Matched and -Mismatched Donor-Recipient Pairs.

Immune reconstitution kinetics and subsequent clinical outcomes in HLA-matched recipients of allogeneic stem cell transplantation (SCT) are variable and difficult to predict. Considering SCT as a dynamical system may allow sequence differences across the exomes of the transplant donors and recipients to be used to simulate an alloreactive T cell response, which may allow better clinical outcome prediction. To accomplish this, whole exome sequencing was performed on 34 HLA-matched SCT donor-recipient pairs (DRPs) and the nucleotide sequence differences translated to peptides. The binding affinity of the peptides to the relevant HLA in each DRP was determined. The resulting array of peptide-HLA binding affinity values in each patient was considered as an operator modifying a hypothetical T cell repertoire vector, in which each T cell clone proliferates in accordance with the logistic equation of growth. Using an iterating system of matrices, each simulated T cell clone's growth was calculated with the steady-state population being proportional to the magnitude of the binding affinity of the driving HLA-peptide complex. Incorporating competition between T cell clones responding to different HLA-peptide complexes reproduces a number of features of clinically observed T cell clonal repertoire in the simulated repertoire, including sigmoidal growth kinetics of individual T cell clones and overall repertoire, Power Law clonal frequency distribution, increase in repertoire complexity over time with increasing clonal diversity, and alteration of clonal dominance when a different antigen array is encountered, such as in SCT. The simulated, alloreactive T cell repertoire was markedly different in HLA-matched DRPs. The patterns were differentiated by rate of growth and steady-state magnitude of the simulated T cell repertoire and demonstrate a possible correlation with survival. In conclusion, exome wide sequence differences in DRPs may allow simulation of donor alloreactive T cell response to recipient antigens and may provide a quantitative basis for refining donor selection and titration of immunosuppression after SCT.

Comparison of Lactobacillus crispatus isolates from Lactobacillus-dominated vaginal microbiomes with isolates from microbiomes containing bacterial vaginosis-associated bacteria.

Vaginal lactobacilli can inhibit colonization by and growth of other bacteria, thereby preventing development of bacterial vaginosis (BV). Amongst the lactobacilli, Lactobacillus crispatus appears to be particularly effective at inhibiting growth of BV-associated bacteria. Nonetheless, some women who are colonized with this species can still develop clinical BV. Therefore, we sought to determine whether strains of L. crispatus that colonize women with lactobacilli-dominated vaginal microbiomes are distinct from strains that colonize women who develop BV. The genomes of L. crispatus isolates from four women with lactobacilli-dominated vaginal microbiomes ( <1% 16S rRNA reads above threshold from genera other than Lactobacillus) and four women with microbiomes containing BV-associated bacteria (>12% 16S rRNA reads from bacterial taxa associated with BV) were sequenced and compared. Lactic acid production by the different strains was quantified. Phage induction in the strains was also analysed. There was considerable genetic diversity between strains, and several genes were exclusive to either the strains from Lactobacillus-dominated microbiomes or those containing BV-associated bacteria. Overall, strains from microbiomes dominated by lactobacilli did not differ from strains from microbiomes containing BV-associated bacteria with respect to lactic acid production. All of the strains contained multiple phage, but there was no clear distinction between the presence or absence of BV-associated bacteria with respect to phage-induced lysis. Genes found to be exclusive to the Lactobacillus-dominated versus BV-associated bacteria-containing microbiomes could play a role in the maintenance of vaginal health and the development of BV, respectively.

The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies.

BACKGROUND: Characterizing microbial communities via next-generation sequencing is subject to a number of pitfalls involving sample processing. The observed community composition can be a severe distortion of the quantities of bacteria actually present in the microbiome, hampering analysis and threatening the validity of conclusions from metagenomic studies. We introduce an experimental protocol using mock communities for quantifying and characterizing bias introduced in the sample processing pipeline. We used 80 bacterial mock communities comprised of prescribed proportions of cells from seven vaginally-relevant bacterial strains to assess the bias introduced in the sample processing pipeline. We created two additional sets of 80 mock communities by mixing prescribed quantities of DNA and PCR product to quantify the relative contribution to bias of (1) DNA extraction, (2) PCR amplification, and (3) sequencing and taxonomic classification for particular choices of protocols for each step. We developed models to predict the "true" composition of environmental samples based on the observed proportions, and applied them to a set of clinical vaginal samples from a single subject during four visits. RESULTS: We observed that using different DNA extraction kits can produce dramatically different results but bias is introduced regardless of the choice of kit. We observed error rates from bias of over 85% in some samples, while technical variation was very low at less than 5% for most bacteria. The effects of DNA extraction and PCR amplification for our protocols were much larger than those due to sequencing and classification. The processing steps affected different bacteria in different ways, resulting in amplified and suppressed observed proportions of a community. When predictive models were applied to clinical samples from a subject, the predicted microbiome profiles were better reflections of the physiology and diagnosis of the subject at the visits than the observed community compositions. CONCLUSIONS: Bias in 16S studies due to DNA extraction and PCR amplification will continue to require attention despite further advances in sequencing technology. Analysis of mock communities can help assess bias and facilitate the interpretation of results from environmental samples.

Identification of a gene in Mycoplasma hominis associated with preterm birth and microbial burden in intraamniotic infection.

OBJECTIVE: Microbial invasion of the amniotic cavity is associated with spontaneous preterm labor and adverse pregnancy outcome, and Mycoplasma hominis often is present. However, the pathogenic process by which M hominis invades the amniotic cavity and gestational tissues, often resulting in chorioamnionitis and preterm birth, remains unknown. We hypothesized that strains of M hominis vary genetically with regards to their potential to invade and colonize the amniotic cavity and placenta. STUDY DESIGN: We sequenced the entire genomes of 2 amniotic fluid isolates and a placental isolate of M hominis from pregnancies that resulted in preterm births and compared them with the previously sequenced genome of the type strain PG21. We identified genes that were specific to the amniotic fluid/placental isolates. We then determined the microbial burden and the presence of these genes in another set of subjects from whom samples of amniotic fluid had been collected and were positive for M hominis. RESULTS: We identified 2 genes that encode surface-located membrane proteins (Lmp1 and Lmp-like) in the sequenced amniotic fluid/placental isolates that were truncated severely in PG21. We also identified, for the first time, a microbial gene of unknown function that is referred to in this study as gene of interest C that was associated significantly with bacterial burden in amniotic fluid and the risk of preterm delivery in patients with preterm labor. CONCLUSION: A gene in M hominis was identified that is associated significantly with colonization and/or infection of the upper reproductive tract during pregnancy and with preterm birth.

Skin-to-Skin Care and the Development of the Preterm Infant Oral Microbiome.

OBJECTIVE: The oral cavity represents an initial entry way for oral and gut indigenous colonization. Skin-to-skin (STS) care, in which the mother holds the diaper clad naked preterm (PT) infant between her breasts, is associated with improved digestive function, decreased stress, and improved survival. This study evaluated the development of oral microbial colonization repertoires and health characteristics in PT infants with or without STS exposure. METHODS: Saliva from 42 PT infants (<32 weeks of gestation at birth) was collected prospectively at 1 month and/or at discharge. High-throughput 16S rRNA sequencing identified microbial diversity and prevalence of bacterial signatures correlated with clinical STS or non-STS care. RESULTS: Corrected for gestational age (CGA) at sampling, bacterial taxa demonstrated increased Streptococcus as a signature of oral repertoire maturation. STS was associated with increased Streptococcus (p < 0.024), while non-STS was associated with greater Corynebacterium (p < 0.023) and Pseudomonas (p < 0.019) in infants ≤ 32 weeks CGA. In infants > 32 weeks CGA, Neisseria and Acinetobacter were more prevalent, 50 vs. 16.7% and 40 vs. 0%, respectively. STS care was associated with shorter hospitalization (p < 0.039). CONCLUSION: STS care during earlier gestation was associated with a distinct microbial pattern and an accelerated pace of oral microbial repertoire maturity.

Whole exome sequencing to estimate alloreactivity potential between donors and recipients in stem cell transplantation.

Whole exome sequencing (WES) was performed on stem cell transplant donor-recipient (D-R) pairs to determine the extent of potential antigenic variation at a molecular level. In a small cohort of D-R pairs, a high frequency of sequence variation was observed between the donor and recipient exomes independent of human leucocyte antigen (HLA) matching. Nonsynonymous, nonconservative single nucleotide polymorphisms were approximately twice as frequent in HLA-matched unrelated, compared with related D-R pairs. When mapped to individual chromosomes, these polymorphic nucleotides were uniformly distributed across the entire exome. In conclusion, WES reveals extensive nucleotide sequence variation in the exomes of HLA-matched donors and recipients.

Differences in vaginal microbiome in African American women versus women of European ancestry.

Women of European ancestry are more likely to harbour a Lactobacillus-dominated microbiome, whereas African American women are more likely to exhibit a diverse microbial profile. African American women are also twice as likely to be diagnosed with bacterial vaginosis and are twice as likely to experience preterm birth. The objective of this study was to further characterize and contrast the vaginal microbial profiles in African American versus European ancestry women. Through the Vaginal Human Microbiome Project at Virginia Commonwealth University, 16S rRNA gene sequence analysis was used to compare the microbiomes of vaginal samples from 1268 African American women and 416 women of European ancestry. The results confirmed significant differences in the vaginal microbiomes of the two groups and identified several taxa relevant to these differences. Major community types were dominated by Gardnerella vaginalis and the uncultivated bacterial vaginosis-associated bacterium-1 (BVAB1) that were common among African Americans. Moreover, the prevalence of multiple bacterial taxa that are associated with microbial invasion of the amniotic cavity and preterm birth, including Mycoplasma, Gardnerella, Prevotella and Sneathia, differed between the two ethnic groups. We investigated the contributions of intrinsic and extrinsic factors, including pregnancy, body mass index, diet, smoking and alcohol use, number of sexual partners, and household income, to vaginal community composition. Ethnicity, pregnancy and alcohol use correlated significantly with the relative abundance of bacterial vaginosis-associated species. Trends between microbial profiles and smoking and number of sexual partners were observed; however, these associations were not statistically significant. These results support and extend previous findings that there are significant differences in the vaginal microbiome related to ethnicity and demonstrate that these differences are pronounced even in healthy women.

The association of maternal factors with the neonatal microbiota and health.

The human microbiome plays a crucial role in human health. However, the influence of maternal factors on the neonatal microbiota remains obscure. Herein, our observations suggest that the neonatal microbiotas, particularly the buccal microbiota, change rapidly within 24-48 h of birth but begin to stabilize by 48-72 h after parturition. Network analysis clustered over 200 maternal factors into thirteen distinct groups, and most associated factors were in the same group. Multiple maternal factor groups were associated with the neonatal buccal, rectal, and stool microbiotas. Particularly, a higher maternal inflammatory state and a lower maternal socioeconomic position were associated with a higher alpha diversity of the neonatal buccal microbiota and beta diversity of the neonatal stool microbiota was influenced by maternal diet and cesarean section by 24-72 h postpartum. The risk of admission of a neonate to the newborn intensive care unit was associated with preterm birth as well as higher cytokine levels and probably higher alpha diversity of the maternal buccal microbiota.

Antibody Response to the Sneathia vaginalis Cytopathogenic Toxin A during Pregnancy.

Sneathia vaginalis is a Gram-negative vaginal species that is associated with pregnancy complications. It produces cytopathogenic toxin A (CptA), a pore-forming toxin. To determine whether CptA is expressed in vivo and to examine the mucosal Ab response to the toxin, we examined human midvaginal swab samples obtained during pregnancy for IgM, IgA, and IgG Abs with CptA affinity. This subcohort study included samples from 93 pregnant people. S. vaginalis relative abundance was available through 16S rRNA survey. There were 22 samples from pregnancies that resulted in preterm birth in which S. vaginalis relative abundance was <0.005%, 22 samples from pregnancies that resulted in preterm birth with S. vaginalis ≥0.005%, 24 samples from pregnancies that resulted in term birth with S. vaginalis <0.005%, and 25 samples from pregnancies that resulted in term birth with S. vaginalis ≥0.005%. IgM, IgA, and IgG with affinity for CptA were assessed by ELISA. The capacity for the samples to neutralize CptA was quantified by hemolysis assay. All three Ab isotypes were detectable within different subsets of the samples. There was no significant association between relative abundance of S. vaginalis and the presence of any Ab isotype. The majority of vaginal swab samples containing detectable levels of anti-CptA Abs neutralized the hemolytic activity of CptA, with the strongest correlation between IgA and neutralizing activity. These results demonstrate that S. vaginalis produces CptA in vivo and that CptA is recognized by the host immune defenses, resulting in the production of Abs with toxin-neutralizing ability.

Phase variable colony variants are conserved across Gardnerella spp. and exhibit different virulence-associated phenotypes.

The Gardnerella genus, comprising at least 13 species, is associated with the polymicrobial disorder bacterial vaginosis (BV). However, the details of BV pathogenesis are poorly defined, and the contributions made by individual species, including Gardnerella spp., are largely unknown. We report here that colony phenotypes characterized by size (large and small) and opacity (opaque and translucent) are phase variable and are conserved among all tested Gardnerella strains, representing at least 10 different species. With the hypothesis that these different variants could be an important missing piece to the enigma of how BV develops in vivo, we characterized their phenotypic, proteomic, and genomic differences. Beyond increased colony size, large colony variants showed reduced vaginolysin secretion and faster growth rate relative to small colony variants. The ability to inhibit the growth of Neisseria gonorrhoeae and commensal Lactobacillus species varied by strain and, in some instances, differed between variants. Proteomics analyses indicated that 127-173 proteins were differentially expressed between variants. Proteins with increased expression in large variants of both strains were associated with amino acid and protein synthesis and protein folding, whereas those increased in small variants were related to nucleotide synthesis, phosphate transport, ABC transport, and glycogen breakdown. Furthermore, whole genome sequencing analyses revealed an abundance of genes associated with variable homopolymer tracts, implicating slipped strand mispairing in Gardnerella phase variation and illuminating the potential for previously unrecognized heterogeneity within clonal populations. Collectively, these results suggest that phase variants may be primed to serve different roles in BV pathogenesis.IMPORTANCEBacterial vaginosis is the most common gynecological disorder in women of childbearing age. Gardnerella species are crucial to the development of this dysbiosis, but the mechanisms involved in the infection are not understood. We discovered that Gardnerella species vary between two different forms, reflected in bacterial colony size. A slow-growing form makes large amounts of the toxin vaginolysin and is better able to survive in human cervix tissue. A fast-growing form is likely the one that proliferates to high numbers just prior to symptom onset and forms the biofilm that serves as a scaffold for multiple BV-associated anaerobic bacteria. Identification of the proteins that vary between different forms of the bacteria as well as those that vary randomly provides insight into the factors important for Gardnerella infection and immune avoidance.

Clostridium scindens: a human gut microbe with a high potential to convert glucocorticoids into androgens.

Clostridium scindens American Type Culture Collection 35704 is capable of converting primary bile acids to toxic secondary bile acids, as well as converting glucocorticoids to androgens by side-chain cleavage. The molecular structure of the side-chain cleavage product of cortisol produced by C. scindens was determined to be 11ß-hydroxyandrost-4-ene-3,17-dione (11ß-OHA) by high-resolution mass spectrometry, (1)H and (13)C NMR spectroscopy, and X-ray crystallography. Using RNA-Seq technology, we identified a cortisol-inducible (≈ 1,000-fold) operon (desABCD) encoding at least one enzyme involved in anaerobic side-chain cleavage. The desC gene was cloned, overexpressed, purified, and found to encode a 20α-hydroxysteroid dehydrogenase (HSDH). This operon also encodes a putative "transketolase" (desAB) hypothesized to have steroid-17,20-desmolase/oxidase activity, and a possible corticosteroid transporter (desD). RNA-Seq data suggests that the two-carbon side chain of glucocorticords may feed into the pentose-phosphate pathway and are used as a carbon source. The 20α-HSDH is hypothesized to function as a metabolic "rheostat" controlling rates of side-chain cleavage. Phylogenetic analysis suggests this operon is rare in nature and the desC gene evolved from a gene encoding threonine dehydrogenase. The physiological effect of 11ß-OHAD on the host or other gut microbes is currently unknown.

Endosymbiosis in trypanosomatids: the genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers.

BACKGROUND: Trypanosomatids of the genera Angomonas and Strigomonas live in a mutualistic association characterized by extensive metabolic cooperation with obligate endosymbiotic Betaproteobacteria. However, the role played by the symbiont has been more guessed by indirect means than evidenced. Symbiont-harboring trypanosomatids, in contrast to their counterparts lacking symbionts, exhibit lower nutritional requirements and are autotrophic for essential amino acids. To evidence the symbiont's contributions to this autotrophy, entire genomes of symbionts and trypanosomatids with and without symbionts were sequenced here. RESULTS: Analyses of the essential amino acid pathways revealed that most biosynthetic routes are in the symbiont genome. By contrast, the host trypanosomatid genome contains fewer genes, about half of which originated from different bacterial groups, perhaps only one of which (ornithine cyclodeaminase, EC:4.3.1.12) derived from the symbiont. Nutritional, enzymatic, and genomic data were jointly analyzed to construct an integrated view of essential amino acid metabolism in symbiont-harboring trypanosomatids. This comprehensive analysis showed perfect concordance among all these data, and revealed that the symbiont contains genes for enzymes that complete essential biosynthetic routes for the host amino acid production, thus explaining the low requirement for these elements in symbiont-harboring trypanosomatids. Phylogenetic analyses show that the cooperation between symbionts and their hosts is complemented by multiple horizontal gene transfers, from bacterial lineages to trypanosomatids, that occurred several times in the course of their evolution. Transfers occur preferentially in parts of the pathways that are missing from other eukaryotes. CONCLUSION: We have herein uncovered the genetic and evolutionary bases of essential amino acid biosynthesis in several trypanosomatids with and without endosymbionts, explaining and complementing decades of experimental results. We uncovered the remarkable plasticity in essential amino acid biosynthesis pathway evolution in these protozoans, demonstrating heavy influence of horizontal gene transfer events, from Bacteria to trypanosomatid nuclei, in the evolution of these pathways.

Microdiversity of the Vaginal Microbiome is Associated with Preterm Birth.

Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality. The vaginal microbiome has been associated with PTB, yet the mechanisms underlying this association are not fully understood. Understanding microbial genetic adaptations to selective pressures, especially those related to the host, may yield new insights into these associations. To this end, we analyzed metagenomic data from 705 vaginal samples collected longitudinally during pregnancy from 40 women who delivered preterm spontaneously and 135 term controls from the Multi-Omic Microbiome Study-Pregnancy Initiative (MOMS-PI). We find that the vaginal microbiome of pregnancies that ended preterm exhibits unique genetic profiles. It is more genetically diverse at the species level, a result which we validate in an additional cohort, and harbors a higher richness and diversity of antimicrobial resistance genes, likely promoted by transduction. Interestingly, we find that Gardnerella species, a group of central vaginal pathobionts, are driving this higher genetic diversity, particularly during the first half of the pregnancy. We further present evidence that Gardnerella spp. undergoes more frequent recombination and stronger purifying selection in genes involved in lipid metabolism. Overall, our results reveal novel associations between the vaginal microbiome and PTB using population genetics analyses, and suggest that evolutionary processes acting on the vaginal microbiome may play a vital role in adverse pregnancy outcomes such as preterm birth.

Characteristics of vaginal microbes and classification of the vaginal microbiome.

Background: The vaginal microbiome (VMB) has been classified into several discrete community state types, some of which have been associated with adverse human health conditions. However, the roles of the many vaginal bacteria in modulating the VMB and health remain unclear. Methods: The associations among the vaginal taxa and other vaginal taxa, the vaginal pH, and the host gene expression responses were determined by calculating the correlation among the relative abundance of the vaginal taxa, the association between the vaginal pH and the predominant taxon in the VMB, and the correlation between the relative abundance of the vaginal taxa and human gene expression at the transcriptional level, respectively. Using these associations, an alternative more informative method, the biological vagitype (BVT), is proposed to classify community state types of the VMB. Findings: Most Lactobacillus spp., with the exception of Lactobacillus iners , show significant correlations with host gene expression profiles and negative associations with dysbiosis-associated vaginal taxa. Many non- Lactobacillus spp. exhibit varied correlations with Lactobacillus spp., the vaginal pH, and host gene expression. Compared to other dysbiotic taxa, including Candidatus Lachnocurva vaginae, Gardnerella vaginalis has a stronger positive correlation with vaginal pH and a stronger negative correlation with Lactobacillus spp. Most dysbiosis-associated taxa are associated with stress responses of the host at the transcriptional level, but the genus Mycoplasma has a uniquely strong positive correlation with host immune responses. The association between BVTs of the VMBs and host characteristics, e.g., race/ethnicity, microbial infection, smoking, antibiotics, high blood pressure, economic state, diet, and others, was examined. The BVT classification method improved overall performance in associating specific vaginal microbial populations with host characteristics and phenotypes. Interpretation: This study sheds light on the biological characteristics of the vaginal microbiota, including some less abundant or still unculturable taxa. Since the BVT method was established based on these biological characteristics, the classification outcome of the VMB may have more clinical relevance. Because the BVT method performs better in associating specific vaginal community types with diseases, e.g., bacterial vaginosis and gonorrhea, it could be beneficial for the predictive modeling of adverse health. Funding: This work was supported by grants [UH3AI083263, U54HD080784, and R01HD092415] from the National Institutes of Health; and support from the [GAPPS BMGF PPB] grant from the Global Alliance to Prevent Prematurity and Stillbirth. We would also like to thank the Office of Research on Women's Health at NIH for their generous support. Research in context: Evidence before this study: The vaginal microbiome (VMB) refers to the community of microorganisms in the female lower reproductive tract. The VMB is often a simple ecosystem dominated by a single species. The most predominant bacteria in the VMB include several Lactobacillus species and two non- Lactobacillus species, i.e., Candidatus Lachnocurva vaginae and Gardnerella vaginalis. Lactobacillus species produce lactic acid to lower the vaginal pH and inhibit the growth of disease-associated bacteria. Thus, the predominance of protective Lactobacilli, i.e., L. crispatus, L. jensenii , and L. gasseri , in the VMB is associated with overall vaginal health. However, the role of L. iners in promoting a healthy vaginal ecosystem is less clear. Actually, the biological and health relevance of many bacteria in the female lower reproductive tract is largely unknown. Some bacteria have low relative abundances, e.g., Peptostreptococcus and Coriobacteriaceae spp.; and others are not yet culturable, e.g., Candidatus Lachnocurva vaginae and BVAB TM7. When abundance of a taxon is low, its association with a host characteristic is a challenge. Previous methods to classify the VMB were based simply on their microbial compositions, and the biological characteristics of the vaginal bacteria were largely ignored. Thus, classification of these VMBs into biologically relevant community types, as described herein, should be helpful in determining their relevance to women's reproductive health. Added value of this study: This study examines three biological characteristics of bacteria in the VMB, i.e., the associations among different bacterial taxa, the vaginal pH, and the host response. Based on these three characteristics, the influence of these bacteria, particularly low abundant and unculturable bacteria, on vaginal health is evaluated. L. iners seems to be neutral in maintaining overall vaginal health. Gardnerella vaginalis is apparently more easily inhibited by Lactobacillus spp. than Candidatus Lachnocurva vaginae because of its stronger positive correlation with vaginal pH and negative correlation with Lactobacillus . The genus of Mycoplasma has a unique positive correlation with local immune responses, implying a role for Mycoplasma in promoting inflammation. Compared with previous methods to classify the VMB, a new method, considering the above three biological characteristics of bacteria in the VMB, has been established. The new method performs better in associating specific vaginal communities with host characteristics and phenotypes; e.g., bacterial vaginosis and gonorrhea. Implications of all the available evidence: Accurate biological classification of the VMB is fundamental for assessing its impact on women's health. Our classification scheme represents a step further toward that correct classification, eventually leading to new strategies for clinical assessment of the potential use of the VMB to diagnose or predict women's reproductive health.